Philips A V, Coleman M S, Maskos K, Barkley M D
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Biochemistry. 1989 Mar 7;28(5):2040-50. doi: 10.1021/bi00431a012.
Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.
腺苷脱氨酶是一种对免疫能力至关重要的嘌呤补救酶，采用时间分辨荧光光谱法对其进行了研究。这种含有四个色氨酸的蛋白质的非均匀发射被分离为三个寿命组分：τ1 = 1纳秒，τ2 = 2.2纳秒，发射峰在约330纳米处，以及τ3 = 6.3纳秒，发射峰在约340纳米处。用极性和非极性荧光猝灭剂探测色氨酸发射的溶剂可及性。丙烯酰胺、碘化物和三氯乙醇猝灭了所有三个组分的发射。丙烯酰胺猝灭导致组分3的衰变相关光谱发生蓝移。基态类似物酶抑制剂嘌呤核苷猝灭了与组分2相关的发射，而过渡态类似物抑制剂脱氧助间型霉素猝灭了组分2和3的发射。抑制剂结合引起的猝灭对衰变相关光谱的寿命或发射峰没有影响。这些观察结果可用一个包含四个色氨酸环境的简单模型来解释。对酶 - 抑制剂复合物的猝灭研究表明，腺苷脱氨酶在结合基态和过渡态类似物抑制剂时会发生不同的蛋白质构象变化。结果与该酶中局部结构改变一致。
